Poxviruses are the largest known animal viruses with approximately 200 distinct genes (Moss, In: Fields Virology, ed. by Knipe and Howley, Philadelphia: Lippincott Williams & Wilkins, 2001, pp. 2849-2883). They are DNA viruses that replicate entirely in the cytoplasm. Thus, a subset of their gene products carries out the functions that are essential for the viruses to be independent of the host-cell nucleus. The other viral gene products use or modulate a wide array of host-cell and immune-system processes.
Poxviruses infect most vertebrates and invertebrates causing a variety of diseases of veterinary and medical importance. The Poxviridae family has two main subfamilies, the chordopoxvirinae, which infect vertebrates, and the entomopoxvirinae, which infect insects. The chordopoxviruses include the genera Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Mollusciposvirus, and Yatapoxvirus. Each of the chordopoxviruses has a restricted and specific host array. Humans are the sole hosts of two poxviruses, variola virus (smallpox virus) and molluscum contagiosum virus; however many members of Orthopoxvirus, Parapoxvirus, and Yatapoxvirus are zoonotic, i.e., can infect both animals and humans. Vaccinia virus is the virus used in the variola virus vaccine, and it is widely used as a model poxvirus in the laboratory. Variola virus and vaccinia virus are members of the Orthopoxvirus genus.
Smallpox virus (variola major) has the potential to be used as a weapon. The virus is contagious, easy to store, and infection is fatal in 30-40% of unimmunized individuals (Davis et al., Microbiology, Hagerstown, Md.: Harper & Row, 1980, pp. 1077-1093; Harrison et al., Proc. Natl. Acad. Sci. USA, 101(31): 11178-11192, 2004). Importantly, more than half the world's population has never been vaccinated against this virus and essentially no one has been re-immunized for at least 20 years. Efforts to resume immunization and re-immunization have foundered. A drug that inhibited the growth of variola should prevent and cure acute smallpox infection even in unimmunized individuals. To this end, efforts have been made to develop nucleoside analogs as anti-poxvirus drugs (Bray et al., J. Infect. Dis., 181: 10-19, 2000; De Clercq, Clin. Microbiol. Rev., 14: 382-397, 2001; Keith et al., Antimicrob. Agents Chemother., 47: 2193-2198, 2003). One promising drug currently available is Cidofovir, a cytosine analog (Keith et al., Antimicrob. Agents Chemother., 47: 2193-2198, 2003). It shows considerable anti-poxvirus activity in animals but cannot be administered orally.
The poxviruses, including variola and vaccinia viruses, encode two protein kinases, B1 and F10 (Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91: 7653-7657, 1994; Lin et al., J. Virol., 66: 2717-2723, 1992; Traktman et al., J. Biol. Chem., 264: 21458-21461, 1989). Both kinases are essential for vaccinia virus growth. Temperature-sensitive mutations in either the B1 or F10 genes prevent vaccinia virus multiplication at the non-permissive temperature (Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91: 7653-7657, 1994; Traktman et al., J. Biol. Chem., 264: 21458-21461, 1989). Additionally, vaccinia virus F10 catalytic activity has been shown to be required for virus growth (Szajner et al., J. Virol., 78: 257-265, 2004). Substances that inhibit the enzymatic activity of either or both poxvirus protein kinases are needed to increase and diversify the arsenal of anti-poxviral drugs.
One consideration in the discovery of viral protein kinase inhibitors is cross-reactivity with protein kinases normally expressed in host cells. Human cells, for example, express hundreds of protein kinases that often have important functions in the regulation of cell growth and metabolism. An anti-viral drug that coincidentally inhibits important cellular protein kinases may have adverse side effects for the host cell and/or organism; however, this is certainly not always the case. Several high-affinity inhibitors of cellular protein kinases have proved to be both useful in treatment of a number of human malignancies and to exhibit acceptable toxicity. Gleevec (Novartis), an inhibitor of Abl, the platelet-derived growth factor receptor, and the Kit receptor is efficacious in chronic myeloid leukemia (Druker et al., N. Engl. J. Med., 344: 1031-1037, 2001; Joensuu et al., N. Engl. J. Med., 344: 1052-1056, 2001), gastrointestinal stromal tumor (Joensuu et al., N. Engl. J. Med., 344: 1052-1056, 2001), and hypereosinophilia syndrome (Schaller and Burkland, Med. Gen. Med., 3: 9, 2001). Iressa (AstraZeneca), an inhibitor of the epidermal growth factor receptor, is valuable in treatment of a subset of small cell lung tumors (Han et al., J. Clin. Oncol., 23: 2493-2501, 2005; Mitsudomi et al., J. Clin. Oncol., 23: 2513-2520, 2005). The foregoing drugs are tolerated well even when used chronically.
Advantageously, a poxvirus protein kinase inhibitor would only need to be used acutely to treat or prevent variola infection. The virus life cycle is less than 24 hours and infection with smallpox virus lasts approximately two weeks in humans (Davis et al., Microbiology, Hagerstown, Md.: Harper & Row, 1980, pp. 1077-1093). Therefore, even if a viral protein kinase inhibitor cross-reacted to some extent with host cell protein kinases, any side effects of the inhibitor may be short lived and clinically acceptable.
New drugs are needed to combat the multitude of diseases caused by poxviruses, especially those poxviruses that could be used as bioterrorism agents, such as variola virus.